Expression Inhibitor of Inflammation Promoting Factor, Screening Method for Active Ingredient Thereof, Expression Cassette Useful for Said Method, Diagnostic Agent and Diagnosis Method
a technology expression inhibitor, which is applied in the field of expression inhibitor of inflammation promoting factor, and the use of expression cassettes for said methods, can solve the problems of unelucidated protein functions
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example 1a
[0298]For the purpose of discovering an IL-6 post-transcriptional regulatory factor, screening was carried out. The schema is shown in FIG. 1A. Concretely, the following procedures were carried out. First, a reporter vector in which a luciferase ORF and an IL-6 3′UTR (SEQ ID NO: 1) were arranged downstream from a SV40 promoter in this order as observed from the 5′ side was produced. The reporter vector and an expression vector for each of various genes were transfected into a HEK293T cell on a 384-well plate using a transfection reagent (Fugene HD, manufactured by Promega Corporation). 48 hours after the transfection, the luciferase activity in each well was measured. A measurement value thus obtained was compared with a measurement value of a control (i.e., a sample in which an empty vector was introduced as an expression vector), and an expression vector of which the measurement value was changed compared with the control was screened (primary screening). About 100 gene expression...
example 1b
[0300]The reporter vector produced in Example 1A or a control reporter vector which was produced by removing a human IL-6 3′UTR from the reporter vector was introduced into a HEK 293 T cell together with a RBMS2 expression vector or an empty vector. 48 hours after the transfection, a luciferase activity was measured. The results are shown in FIG. 1B.
[0301]As shown in FIG. 1B, RBMS2 increased a luciferase activity in an IL-6 3′UTR-dependent manner.
example 1c
[0302]siRNA for RBMS2 (Human RBMS2-1 (Thermo Fisher Scientific (Ambion), Silencer Select, siRNA ID No. s11867, SEQ ID NO: 8)), or Human RBMS2-2 (Thermo Fisher Scientific (Ambion), Silencer Select, siRNA ID No. s11868, SEQ ID NO: 9), or control siRNA (Negative control (Thermo Fisher Scientific (Ambion), Silencer Select Negative Control No. 1 siRNA, Product number: 4390843)) was introduced into a MDA-MB-231 cell. 48 hours after the introduction, IL-1β was added to a culture medium (final concentration: 20 ng / ml), and the cell was collected 3 hours after the addition. In this regard, a MDA-MB-231 cell can constantly produce IL-6, and can express IL-6 in a larger amount / level upon the stimulation with IL-1β. cDNA was prepared from the cell, and the expression amount / levels of RBMS2 mRNA (PCR primer: Forward: SEQ ID NO: 12, Reverse: SEQ ID NO: 13) and IL-6 mRNA (PCR primer: Forward: SEQ ID NO: 14, Reverse: SEQ ID NO: 15) were measured by quantitative PCR. The results are shown in FIG. 1C...
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